Johan T. Burger

Deep sequencing analysis of viruses infecting grapevines: Virome of a vineyard

Double stranded RNA, isolated from 44 pooled randomly selected vines from a diseased South African vineyard, has been used in a deep sequencing analysis to build a census of the viral population. The dsRNA was sequenced in an unbiased manner using the sequencing-by-synthesis technology offered by the Illumina Genome Analyzer II and yielded 837 megabases of metagenomic sequence data. Four known viral pathogens were identified. It was found that Grapevine leafroll-associated virus 3 (GLRaV-3) is the most prevalent species, constituting 59% of the total reads, followed by Grapevine rupestris stem pitting-associated virus and Grapevine virus A. Grapevine virus E, a virus not previously reported in South African vineyards, was identified in the census. Viruses not previously identified in grapevine were also detected. The second most prevalent virus detected was a member of the Chrysoviridae family similar to Penicillium chrysogenum virus. Sequences aligning to two other mycoviruses were also detected.

Grapevine leafroll-associated virus 3

Grapevine leafroll disease (GLD) is one of the most important grapevine viral diseases affecting grapevines worldwide. The impact on vine health, crop yield, and quality is difficult to assess due to a high number of variables, but significant economic losses are consistently reported over the lifespan of a vineyard if intervention strategies are not implemented. Several viruses from the family Closteroviridae are associated with GLD. However, Grapevine leafroll-associated virus 3 (GLRaV-3), the type species for the genus Ampelovirus, is regarded as the most important causative agent. Here we provide a general overview on various aspects of GLRaV-3, with an emphasis on the latest advances in the characterization of the genome. The full genome of several isolates have recently been sequenced and annotated, revealing the existence of several genetic variants. The classification of these variants, based on their genome sequence, will be discussed and a guideline is presented to facilitate future comparative studies. The characterization of sgRNAs produced during the infection cycle of GLRaV-3 has given some insight into the replication strategy and the putative functionality of the ORFs. The latest nucleotide sequence based molecular diagnostic techniques were shown to be more sensitive than conventional serological assays and although ELISA is not as sensitive it remains valuable for high-throughput screening and complementary to molecular diagnostics. The application of next-generation sequencing is proving to be a valuable tool to study the complexity of viral infection as well as plant pathogen interaction. Next-generation sequencing data can provide information regarding disease complexes, variants of viral species, and abundance of particular viruses. This information can be used to develop more accurate diagnostic assays. Reliable virus screening in support of robust grapevine certification programs remains the cornerstone of GLD management.

Complete nucleotide sequence of a South African isolate of grapevine leafroll-associated virus 3 reveals a 5′UTR of 737 nucleotides

Grapevine leafroll-associated virus 3 (GLRaV-3) is the type species of the genus Ampelovirus (family Closteroviridae) [1]. It is an economically important virus that is known to only infect Vitis spp. and that has a negative impact on the wine and table grape industries worldwide. To date, there has been only one report that claims the complete nucleotide sequence of GLRaV-3, by Ling et al. in 2004 (isolate NY-1, AF037268) [2]. In that report the 50 untranslated region (UTR) was found to be 158 nt in length. Here we report that the sequence of GLRaV-3, isolate GP18, has a 50UTR of 737 nt. This extended UTR was found in all other South African isolates of GLRaV-3 that were analysed. Grapevine material (Cabernet Sauvignon) was harvested in the Somerset West wine-producing region in South Africa from a monitored vineyard. RNA of isolate GP18 was extracted from a vine that displayed symptoms for the first time (Pietersen, pers comm). GP18 double-stranded RNA (dsRNA) was extracted from phloem tissue of wooded canes using an adapted cellulose (CF11) column method first described by Hu et al. [3]. RT-PCR was performed with primer sets designed to cover a large portion of the genome (nucleotides 1,835–17,905 of AF037268) in 10 overlapping clones (Fig. 1). Amplicons were cloned and sequenced and a consensus sequence was generated using BioEdit [4]. Primers were not included in the consensus sequence assembly of these overlapping sequences. Poly(A) tailing was performed on dsRNA in an attempt to reach the 50and 30terminal ends (Fig. 1) [5]. The 30-terminus of GP18 was found to be similar to that of the NY-1 isolate. However, we were unable to reach the reported 50-end of the NY-1 isolate and consistently found amplicons that started at +50 nt. After further experimentation with PCR conditions, a range of amplicons were generated that extended beyond the NY-1 sequence’s 50-end. A possible explanation for this inconsistency, using poly(A) tailing, might be the high uracil content in the 50-region upstream of the +50 site in the NY-1 strain that leads to stretches of adenines on the negative strand, which could act as priming sites for the oligo(dT) primer and lead to fragments shorter than the true genomic size. To determine the 50 end of the GP18 genome, total RNA was extracted from grapevine phloem tissue and subjected to RNA ligase-mediated rapid amplification of cDNA Ends (RLM-RACE) using the FirstChoice RLM-RACE kit (Ambion, USA) as per the manufacturer’s instructions. The amplicon generated for the 50-end of the GP18 genome was larger than expected (Fig. 1). It was cloned and four clones sequenced. The 50UTR was found to extend beyond the sequence reported for the NY-1 isolate [2]. The adapter ligation reaction was repeated on the same total RNA treated with calf intestine alkaline phosphatase (CIP) and tobacco acid pyrophosphatase (TAP), and a further five clones were sequenced. All nine clones showed the first 365 nucleotides of the NY-1 sequence and an additional 579 nucleotides upstream of the 50-end. The efficacy of RLM-RACE to determine the 50-termini of multiple ssRNA viruses from total RNA in a single reaction was also investigated. RLM-RACE was performed on total RNA extracted from three grapevine plants (K1, K5, K6) that contained a mixture of viruses (GLRaV-2, -3 and grapevine rupestris stem pitting-associated virus H. J. Maree M.-J. Freeborough J. T. Burger (&) Department of Genetics, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa e-mail: jtb@sun.ac.za 1 The GenBank accession number for the sequence reported in this paper is EU259806.

Grapevine virus A-mediated gene silencing in Nicotiana benthamiana and Vitis vinifera.

Virus-induced gene silencing (VIGS) is an attractive approach for studying gene function. Although the number of virus vectors available for use in VIGS experiments has increased in recent years, most of these vectors are applied in annual or herbaceous plants. The aim of this work was to develop a VIGS vector based on the Grapevine virus A (GVA), which is a member of the genus Vitivirus, family Flexiviridae. The GVA vector was used to silence the endogenous phytoene desaturase (PDS) gene in Nicotiana benthamiana plants. In addition, an Agrobacterium-mediated method for inoculating micropropagated Vitis vinifera cv. Prime plantlets via their roots was developed. Using this method, it was possible to silence the endogenous PDS gene in V. vinifera plantlets. The GVA-derived VIGS vector may constitute an important tool for improving functional genomics in V. vinifera.

Modified Cetyltrimethylammonium bromide method improves robustness and versatility: The benchmark for plant RNA extraction

A wide range of plant RNA extraction methods are available; however, many of these are limited in their application for a diverse range of plant species. With special emphasis on robustness and versatility, we have improved the cetyltrimethylammonium bromide (CTAB) method and isolated high-quality RNA from 16 different plant species. The major modifications made to the protocol described here were a reduction of sample treatment steps and an increase in beta-mercaptoethanol concentration (to 3%) resulting in a robust, rapid and reproducible plant RNA extraction protocol that can be used for a broad range of plant species and tissue types.

Real-time RT-PCR high-resolution melting curve analysis and multiplex RT-PCR to detect and differentiate grapevine leafroll-associated virus 3 variant groups I, II, III and VI

BackgroundGrapevine leafroll-associated virus 3 (GLRaV-3) is the main contributing agent of leafroll disease worldwide. Four of the six GLRaV-3 variant groups known have been found in South Africa, but their individual contribution to leafroll disease is unknown. In order to study the pathogenesis of leafroll disease, a sensitive and accurate diagnostic assay is required that can detect different variant groups of GLRaV-3.MethodsIn this study, a one-step real-time RT-PCR, followed by high-resolution melting (HRM) curve analysis for the simultaneous detection and identification of GLRaV-3 variants of groups I, II, III and VI, was developed. A melting point confidence interval for each variant group was calculated to include at least 90% of all melting points observed. A multiplex RT-PCR protocol was developed to these four variant groups in order to assess the efficacy of the real-time RT-PCR HRM assay.ResultsA universal primer set for GLRaV-3 targeting the heat shock protein 70 homologue (Hsp70h) gene of GLRaV-3 was designed that is able to detect GLRaV-3 variant groups I, II, III and VI and differentiate between them with high-resolution melting curve analysis. The real-time RT-PCR HRM and the multiplex RT-PCR were optimized using 121 GLRaV-3 positive samples. Due to a considerable variation in melting profile observed within each GLRaV-3 group, a confidence interval of above 90% was calculated for each variant group, based on the range and distribution of melting points. The intervals of groups I and II could not be distinguished and a 95% joint confidence interval was calculated for simultaneous detection of group I and II variants. An additional primer pair targeting GLRaV-3 ORF1a was developed that can be used in a subsequent real-time RT-PCR HRM to differentiate between variants of groups I and II. Additionally, the multiplex RT-PCR successfully validated 94.64% of the infections detected with the real-time RT-PCR HRM.ConclusionThe real-time RT-PCR HRM provides a sensitive, automated and rapid tool to detect and differentiate different variant groups in order to study the epidemiology of leafroll disease.

Three genetic grapevine leafroll-associated virus 3 variants identified from South African vineyards show high variability in their 5′UTR

Three genetic variants of grapevine leafroll-associated virus 3 (GLRaV-3) were identified in vineyards of the Western Cape, South Africa. The GLRaV-3 variants were identified by single-strand conformation polymorphism (SSCP) profiles generated from a region amplified in ORF5. ORF5 sequence data confirmed the three genetic variant groups, and a specific SSCP profile was assigned to each variant group. The results of SSCP analysis of this region in ORF5 showed that this method gives a fast and reliable indication of the GLRaV-3 variant status of a plant, which in many instances showed mixed infections. The full genome sequence of one representative of each variant group i.e. isolates 621 (group I), 623 (group II) and PL-20 (group III), was determined by sequencing overlapping cloned fragments of these isolates. The sequences of genomic 5′ ends of these isolates were determined by RLM-RACE. Sequence alignment of the 5′UTRs indicated significant sequence and length variation in this region between the three South African variant groups. Alignment of the Hsp70h and CP gene regions of these isolates with those of isolates from elsewhere in the world, followed by phylogenetic analysis, further supported the presence of three variants of GLRaV-3 in South Africa and the presence of two or three additional variant groups elsewhere in the world.

High-throughput sequencing reveals small RNAs involved in ASGV infection

BackgroundPlant small RNAs (sRNAs) associated with virulent virus infections have been reported by previous studies, while the involvement of sRNAs in latent virus infection remains largely uncharacterised. Apple trees show a high degree of resistance and tolerance to viral infections. We analysed two sRNA deep sequencing datasets, prepared from different RNA size fractions, to identify sRNAs involved in Apple stem grooving virus (ASGV) infection.ResultssRNA analysis revealed virus-derived siRNAs (vsiRNAs) originating from two ASGV genetic variants. A vsiRNA profile for one of the ASGV variants was also generated showing an increase in siRNA production towards the 3′ end of the virus genome. Virus-derived sRNAs longer than those previously analysed were also observed in the sequencing data. Additionally, tRNA-derived sRNAs were identified and characterised. These sRNAs covered a broad size-range and originated from both ends of the mature tRNAs as well as from their central regions. Several tRNA-derived sRNAs showed differential regulation due to ASGV infection. No changes in microRNA, natural-antisense transcript siRNA, phased-siRNA and repeat-associated siRNA levels were observed.ConclusionsThis study is the first report on the apple sRNA-response to virus infection. The results revealed the vsiRNAs profile of an ASGV variant, as well as the alteration of the tRNA-derived sRNA profile in response to latent virus infection. It also highlights the importance of library preparation in the interpretation of high-throughput sequencing data.

Genetic variability within the coat protein gene of Grapevine fanleaf virus isolates from South Africa and the evaluation of RT-PCR, DAS-ELISA and ImmunoStrips as virus diagnostic assays

Grapevine fanleaf virus (GFLV) is responsible for severe fanleaf degeneration in grapevines of all major wine producing regions of the world, including South Africa. In order to successfully control the spread of the virus, specific and reliable diagnostic assays are necessary. The genetic variability of 12 GFLV isolates recovered from naturally infected grapevine plants in the Western Cape region of South Africa were characterised. These samples were subjected to RNA extraction, RT-PCR analysis and sequencing of the coat protein gene (2CCP). Sequence identities between different GFLV isolates from South Africa were between 86-99% and 94-99% at the nucleotide and amino acid levels, respectively. Phylogenetic analysis based on the 2CCP gene sequences showed that the South African isolates form two distinct clades or sub-populations. The specificity and sensitivity of three diagnostic techniques (rapid-direct-one-tube-RT-PCR, DAS-ELISA and ImmunoStrips) for the detection of GFLV were analysed to determine the appropriate diagnostic assay for virus infection. Rapid-direct-one-tube-RT-PCR was found to be the most reliable technique for detection. This is the first report on sequence analysis of full-length 2CCP gene cDNA clones of GFLV isolates from South Africa.

Grapevine leafroll disease and associated viruses: a unique pathosystem.

Grapevine leafroll is the most complex and intriguing viral disease of grapevine (Vitis spp.). Several monopartite closteroviruses (family Closteroviridae) from grapevines have been molecularly characterized, yet their role in disease etiology is not completely resolved. Hence, these viruses are currently designated under the umbrella term of Grapevine leafroll-associated viruses (GLRaVs). This review examines our current understanding of the genetically divergent GLRaVs and highlights the emerging picture of several unique aspects of the leafroll disease pathosystem. A systems biology approach using contemporary technologies in molecular biology, -omics, and cell biology aids in exploring the comparative molecular biology of GLRaVs and deciphering the complex network of host-virus-vector interactions to bridge the gap between genomics and phenomics of leafroll disease. In addition, grapevine-infecting closteroviruses have a great potential as designer viruses to pursue functional genomics and for the rational design of novel disease intervention strategies in this agriculturally important perennial fruit crop.